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,IN3TIcS AND ilOHANI3LS GI PD( AND I mI-

V 4 CATAIMSD AND I zD lTIONB

By D. T. IMORE, E. F. WRAG, and N. J. BAINE

room Queen's University, Belfast, N. Ireland.

Contract Number M2A-91 -591i-EUC-i 686

Final. Technicatl Report. F. No. i.

m I Pc.-'iod: 11st March, 1961 - 30th September, 1962.

I" Te research reported in this document has been

nada possible th-rough the support and sponsorship

if the U.S. Department of Iamy, through its European

Researc' Oif'ice.

1~E 13' 1963

L Ljl 7 ,. .

b

OL"AL Ir; -

,, . copies, .. _

- I

In Part 1, confirmetory evidence has been adduced or the mechanism of lacta-

alzation of methyl eaters of a--toluen6-r-sulphoryl derivatives of aw-diamino-acids

in aqueous solution, which waa described in the Pinal Technioal Report under Contract

DA-91-599-NC-1301. Participation of the sulphonamide-group has been excluded, since

the kinetics of lactasization of methyl y-amino-.--F-methyltoluene-k-sulphonmidobuty-

rate (M!aI), in which ionisation of the sulphonamido group is precluded, are exaotly

parallel to those of the compounds studied earlier.

In Part II, kinetic studies of the action of trypsin on various synthetic sub-

strates have been amplified. The significance of the experimentally observed values

of various kinetic constants is considered theoretically in the light of the probable

three-step mechanism for catalysis by proteolytic enzymes such as trypsin. In the

trypsin-catalysed hydrolysis of esters of a-F-toluene-2-sulphonyl--arginine, aoylation

and deacylation both appear to influence the overall rate of reaction, while in the

case of eaters of a-Lf-benzoyl-k-arginine, deacylation is rate-determining. In

ionizable group, whose apparent pKa and heat of ionization are consistent with those

of an imidazole nucleus, is implicated in the trypsin-catalysed hydrolysis of a-LI-

toluene-g-sulphonyl--arginine mothyl ester. Cholate ion accelerates the trypsin-

catalysed hydrolysis of esters of a-Lj-toluen-.L>-slphoryl-k.arginine and -homoarginine

slightly inhibits the hydrolysis of a-f-benzoyl-L-arginine ethyl ester (B=), Using

substrato concentrations high enough to ensure obedience to zero-order kinetics,

activation constants have been dctermined. Those for the trypsin-catalysed hydrolysis

of the methyl and ethyl esters of a-I-toluene-gI-ulphonyl-,-arginine (TANE and TAn)

converge to practically identical values with increasing cholate-conoentration. This

suggests that in presence of sufficient cholate the acylation step is sufficiently

accelerated so that deacylation of a common intermediate becomes rate-determining.

The same effect is possibly exerted by cholate on the hydrolysis of a-f-toluene--

aulphonyl-k-homoarginine mothyl ester, and supporting evidence is adduced from deter-

mination of apparent % values. a-I1-Toluene-2-sulphonyl-ig-ornithine methyl ester is

slowly hydrolysed by trypsin at pH 7.0 and the kinetics are first-crder with respect

to substrate. There is evidence that this reaction is not completely stereospecifio.

The hydrolysis is appreciably accelerated by cholate with an attendent decrease in the

apparent energy of activation for the reaction.

In Part III, we report a study of the kinetics of thrombin-catalysed reactions.

Using the Ehr(npreis and Scheraga unit (. Bil. Chem., 1957, U 1%.3) as a means of

expressing thrombin activity, it has been ahown that the velocity of a thrombin-

catalysed reaction is proportional to onzyme concentration. In contrast to

-2-

trypsin-oatelysed reactions, the velocity of hydrolysis of TAME by thrombin is decreased

by sodium or potassium chloride. Like trypsin-catalysed reactions, on the other hand,

the rate-determining step for the hydrolysis of esters of L-2-benzoyl--arginine by

thrombin appears to be deacylation. In the case of esters of as--toluene-j>-sulphorWl-

-einine, both acylation and deacylation influence the overall rate of hydrolysis.

The methyl ester of a -toluene-k-sulphocl-k-lysine (TIE) is hydrolysed by thrombin

at a rate similar to that of TAME. Rate oonotants for the thrombin-catalysed hydroly~d

of TAME and BIM do not obey the Arrhenius law. The apparent dissociation constant of

the ionizable group, which is involved in the thrombin-catalysed hydrolysis of TAME, is

rather lower than that associated with the trypsin-catajlysed hydrolysis of this substrete.

Cholate and glycocholate accelerate the thrombin-catalysed hydrolyses of substrates con-

taining the a-A-toluenew-sulphonamide group, but inhibit the hydrolysis of B . The

apparent % values for the TIME- and BAZ-thrombin systems are both decreased by bile

salts. The mechanism by which bile salts modify the kinetics of throubin-catalysed

reactions is probably similar to that postulated for trypsin-catalysed processes.

Non-ionic detergents such as Tween 20, Twceen 40 and Tween 80 accelerate the hydrolysis

of both TAME and BM by thrombin.

PART I. T KINETICS OF LACTAMIZATION OF METHYL ES7M CP SCME m-N-TVWUM-

p-SUWHONL UIVATVE OF ao-DIJDC=IO S IN AQUB SOLUTION.

In Part I of the Final Technical Report under the preoedinp contract

(No. DA-91-591-N JC-1301), it was shown that laotamization of the methyl esters of

G--toluene-2-sulphonyl-J-crnithine (TOE) and y-amino-c-toluene-L-sulphonamido-

butyric acid (AME) proceeded with hydroxyl-ion oatalysis in the pH range 7.8 -

8.5. We proposed the following mechanism for this reaction:

I H3 H

0 rT s.NRd.C Tos.NRI.. Tos.N.I. -C

"-C~e Me

MI (II) (inI)I-

To .NR. - W----.UIWC - 0(iv)

The initial rate of proton release at constant pH, U., was given by the equation:

+ ............. ()

where k is the second-order rate constant for the lactamization process K is the

dissociation constant of the protonated amino-group of the estcr (I), XW is the

ionic product of water, and [S] 0 is the initial concentration of substrate. The

value of K, as evaluated from the application of equation (i) to our experimental

data, was inconsistent with the participation of the sulphonamide group, and such

a mechanism appeared unlikely. In order to obtain definitive evidence on this

point, m have now synthesised methyl y-amino-L-a--methyltoluene-L-sulphonamido-

butyrate (AMTMM) in which ionization of the sulphonanmide group is precluded. This

compound was readily obtained by methylation of y-benzylo3carborlamido-k-a-toluen.-

]-oulphonamidobutyric acid in a manner similar to that described for the synthesis of

sarcosine (Cocker and Lapworth, g. Chem. §2., 1931, 189.; Cocker, . C 8.,

1 937, 1693). Hydrogenation and esterification completed the synthesis. The y-

benzyloxycarbonylamido-group was proved to be unaffected by methylation, since the

acid afforded by hydrogenation yielded nitrogen when treated with nitrous acid in

"-a Van Slyke apparatus. In addition, the ultraviolet spectrum of the J-methyl acid,

like that of fi-, oluen- .ulphs 'lsaeoosine, was identical in acid and alkaline

solution, whereas that of .-amino-k-a-toluene-2-suphonamidobutyrio acid exibited

a pronounced hyerchromic effect at high pH values resulting from ionization of the

aulphonamide grou. The kinetics of laotamization of AM were founC to be very

similar to those of the two substrates previously studied, and our earlier views

concerning the mechanism of lactamization are fully confirmed. Laotamization of

these esters appears not to be subject to general-base catalysis, since neither

pyridine nor imidazole affected the velocity of reaction. More kinetic runs have

been carried out on TOME and ATMU since the last Final Technical Report, and the

complete results are given in Table I and 2.

TAXL I.

Initial rates (I0 .Uo, mole l.1xmin.-') of proton release during lactamization of the

esters (II) (standard errors of the means in parentheses).

R = H, 2=2 R=Me, =2 R=H, =3

PH 20° 250 30o 250 250 300

7.80 0.76(0.01 1.30(0.03 4.4 0.04 1.02 0.01 1.72(0.05 5.25(0.117.90 1.220.06 2.31 0.08 6.62 0. 17 1 .45 0.07 2.98(0.07 6.9070.128.00 1.86 0.03 3.30 0.07 8.80 0.22 2.19 0,4 4.32 .17 11.08 0.258.10 2.53 - + 4.35 0.7 11.34 0.41 3.5 0.08 6.27 0.08 16.22 O.168.20 3.75 0.07 5.31 0.12 15.C o.69 4.700.1 7 9.300.40 23.09 08.30 4.89 A0.17 7.58O 02 2 5 0.33 6.18 0.4 12.07(0.39 30.35 0.778.A 7.23 0.20 9.i8 0.36 27.94 0.93 8.27 0.53 21.39(0.80 44.44 1.328.50 9.980.0491 12.70,30.19 36.450.77, 11.32(0.28 30.4311.07 59.77(4.19

TABE 2.

pl values for the esters (II) and second-order rate constants (10-4k, mole Lun.for their lactamiztion (stanarrd deviations in parentheses).

R =H, =2 R =Me, =2 R =H,n= 3

20P 250 300 25' 250 30

1 ...... 8.72(0.10) 8.47(0.09) 8.42(0.03) 8.66(0.06) 8.97(0.20) 8.69(0.10)1O- 4 k... 1.77(0.18) 1.5(0.13) 3.0(0.0 9) 1.450.09) 4-.66(0.93) 5.13(0.52)

-"

PARTI.m ' T1 KINnTIC. AJ3 (7 T' mPIN-&AI

In Parts II and III of the Final Technical Report under contract M-9i-51-C-

1301, we reviewed the available evidence related to the meohanism of aotion of

trypsin and we presented the results of our kinetic studies using various synthetic

substrates. We reported also that the trypsin-catalysed bydrolyses of the methyl

esters of a-L-toluen-sulphorwl-k-arginine and -- homoarginine (TAME and TaW

respectively) were accelerated in the presence of cholate ions. We do not propose

to review the literature again at length here, and we shall confine ourselves to

presenting the results of our continued studies of trypsin mentioning only those

publications which are pertinent to the development of our concept of the mechanism

of action of trypsin.

Shwert and Eisenberg (. Biol. chem., i91 9, jD, 665) found that, at substrate

concentrations high enough to ensure that zero-order kinetics were obeyed, several

esters of a--benzoyl-j-argininc were hydrolysed by trypsin at identical rates.

The inference to be drawn from these experiments is that a commrn intermediate,

(a-fi-benzoyl-L-argiryl) trypsin, is formed and that the deacylation of this is

rate-determining. The hydroityl group in the serine residue at the active centre

is probably the site of acylation. Strong evidence for such intermediates has

been obtained in the case of chymotrypsin-catalysed reactions (P. Desnuefle in

"The Enzymes", edited by P. D. Boyer, H. Lardy, and K. Myrblick, Academic Press,

N.Y. 1960, Vol. 4, p.93). The formation of cinnamoyl-chymotrypsin as an inter-

mediate in the hydrolysis of esters of cinnamic acid by cklnotrypsin has been

particularly firmly established (Bender & Zerner, j. -a 9=0 12200 196i, U

2391). The hydrolysis of J-.Van-cinnamoylimidazole is catalysed by either trypain

or chymotrypsin at pH 5.2, and the formation of an intermediate ciramoyl-derivative

of the enzyme has been demonstrated in each case (Bender, Kaiser and Zerner, 1. AM.

Chem. AM., 1961, 83, 4656). The kinetic mechanism of action of these enzymes can

thus be summarized in the expression:

RICEk +

R.C0.0 + Enye "~ acyl- -!'-- pm +hzaymesubstrate enzymecomplex (3) °(,.

Both the enzyme-auastrate complex and the acyl-ensymo are assumed to contain acidic

groups which must lose a proton for reaction to proceed. The p% values of these

-6-

aups are not necossarily identical. Indeed, if an imidazole group is implicated,

hydrogen-bonding between the hydroxyl group of the serine residue Fnd imidazole could

occur in the enzyme-substrate complex, but not in the acyl-enzyme. On the other hand,

hydrogen-bonding could occur between the imidazolium group and the carborWl-group in the

acyl-enryme. Consequently, the p% of the group in the enzyme-substrate complex would

be expected to be lower than that of the group in the acyl-enzyme. Such a difference

has been found in the chyotrypsin-catalysed hydrolysis of R-nitrophenyl acetate

(utfreund and Sturtevant, Prec. Nat. Acad. §c., 1956, h, 719). These authcs have

also derived the kinetic uquations which govern the three-stage mechanism outlined above.

When the steady state has been established, the velocity of reaction is given by:, I

V k2 Z [S] [3] (3)

where k2. .. .............. (4)2 K , + T H 4

and k k 3 K2 ......................3

Experimentally an apparent rate constant k;(nl ) end an apparent Michaelis constant

(sapp) are obtained, and these are related to the true rate constants as follows:-

-4. A .4. * ........... %......... (6)

(app) k k3 k + k2 (7)

k2"+k; 3 k2 +k3 k1

The determination of the effect of pH on kl(app) leads to an apparent dissociation oon-

stant, K(,p),wich is a function of the dissociation constants X and K2 and the rats

constants k and k.

K (app) W11Y2 k+ k 32(8

When deacylation of the acyl-enyme is rrte-determining, k2. t)k 3 anid, provided that 9

does not gretly exceed K

Conversely, if the acylation of enzyve is rate-determining, k2 ( , and, provided that

K, does not greatly exceed 12

,(app) V

In general, let k2 ook 3 andk =i R 12Y

Hence, K(app) = + + (n J)

If Z> 1, N) K(app)) 12 and conversely, if m( i, Y "(app) )

-7-

Thus, K(app) always lies between the two dissociation oonstants, but under special

oonditions may approximate to one of them.

We have studied the kinetios of the trypsin-catalysed hydrolysis of the methyl,

ethyl, and cyclohexyl esters of a-f-toluene-2-sulphonyl-k-arrinine (TAME, TAEM and

TAM respectively). By using substrate conoentrations high enough to ensure zero-

order kinetics, we were able to measure k3 (ap) over a range of temperatures and thus

to determine the activation constants. The results are presented in Tables 3 and 4.

Tabl 3

Rate constants at rH 8.4 for the tryesin-catalvsed hydrolysis of derivatives of arainim

andhmL1 D&.

Rate constants (k3(app)) are quoted with standard deviations; the number of deter-

minations is given in parcntheses.

t°C TAR TALI TACI t BLAM

-(sec. (sect (secZ;) (sec.)

15 49.22±2.21 (4) 63.4±-2.-9() 7.086-0.174(4) 2.286 -±0.098(7)

20 67.442.80(4) 85.44;t8.86(20 10, O20.66 (4) 3.042 !0.108(8)

23 85.72t.35 (.)

25 93.0 .73(7) 93.45±o.47(3) 108.6 :4..O (6) %."0.62 (4) 4.ai !0.212(8)

28 10.6 t5.5 (6)

30 124.7 !6.6 (10) 123.8 -*.7 (3) 146.7 8.7 (s) 21.1, .37 (6) 5.33- 0.231(8)

37 176.8 ±7.5 (6) 183.8 ±7.7 (8)

Tabl 4Activation constants at rH 8.. for the trvosin-catalvscd hydrolysis of derivtives

of ine and hoaogrinine.

Activation constints, with standard deviations, were calculated by the method of

weighted leat squares.

Ra(as./nole) a (cens./mole at 250) W (cas./mole/oC at 250) AGIG'Ccsawbe at

TM .0 -+ 10 883.0 10 -19.8 -0.4 14749± 2

TAZ310450 320 9860 320 -16.4 - 1.1 1.762± 2

8700 390 8110 390 -22.0 ±1.3 14679 11

BM12510- 350 11920 350 -13.2 ±1.2 15856 10

BM 9810 70 9220 70 -21.8 ±0.2 16621± 2

-It will be seen that there are small but definite differences between the values

obtained using TAME, TAZE, and TACO1. There are two possible explanations. The

hydrolysis of the acylated trypsin may not be rate-determining and k 3 (aP) mayI I

include contributions from both k2 amd k • Alternatively, the mechanism is more

complex than that outlined above. Bender and Glasson (I. AM. g=. IM., 1960, Z

3336) have obtained strong evidence for the presence of a site in ohymotrypsin wioh

binds water or alcohols, and it is possible that a similar site exists in trypsin.

The hydrolysis of the acylated enzyme may be an intramolecular process involving a

molecule of water bound at a site close to the acyl group. If the molecule of

alcohol, which is formed during the acylation of trypsin by the eater substrnte, oom-

pates with water for this site, the acyl-trypsin intermediates formed from the three

substrates can no longer be regarded as identical.

We pointed out in the previous Final Technical Report that the entropy of acti-

vation for the hydrolysis of MAL'E is more negative than for the hydrolysis of VJME,

suggesting a greater degree of structural rAganisation in the activatee complex.

This may reflect a deformation of the enzyme, an increased binding of solvent or a

more complex distribution of charges over one of the activated complexes involved in

the hydrolytic mechanism. Unfortunately, we have no information so far concerning

the nature of the rate-determining step.

"e kinetic constants for the trypain-catalysed hydrolysis of ea-benzoy1-L-

arginine ethyl ester (BAER) are close to those recorded for the related methyl ester

(DIME) (Schwert and Eisenberg, g. "o .g., 194~9, IM 665) after correction of

the value frr S* reported earlier (Final Technical Report, D -91-59it-E-301). This

supports the postulate that a comeon intermediate (W-Mbenzoyl-k-argirYl)trypsin is

formed and that deacylation of this by water is rate-determining. It also implies

that ethanol and methanol are not appreciably bound in the acylated intermediate.

Determination of k (app) at various 1 values and temperatures has been carried

out using the TAM-trypsin system. We were able to calculate the apparent disso-

ciation constants at the different temperatures (Table 5), K(ap)9 and the apparent

heat of ionisation.

Temperature ("C). PNl

TMJ~ !HME

15 7.770 0.o220 7.688 0 O.04425 7.529 O.OB 6.9N ! 0.037

30 7. - 0.038

The apparent heat of iciasation for the TAN-trypsin system was 7070 - 530 oals./

mole, a value consistent with that far an imidazole nucleus. It is notabie that the

pg. for the HAIM-trypsin is considerably lower than that for the TAME-trypsin system

at the same temperature. Since it is likely that K. the lower' K2 or p "PX2

apparent pKa for the THAM-trypsin system may be an indication that the ratio k 2 /k 3

is much lower for this system than for the TAMI-trypsin system. In other words, the

acylation step may be the main rate-limiting stage in the mechanism. Our results

differ somewhat from those of Ronwin 4oZhin. Bio i Ada 1959, N, 326), but the

conditions were rather different.

We next compared the effect of cholate on the kinetics of the trypsin-catalysed

hydrolyses of TAIM and TA at high substrate oonoentrations. Unfortunately a

similar investigation using TAMH was precluded, since addition of cholate to an

aqueous solution of this substrate gave a precipitate. The hydrolysis of both sub-

strates was accelerated by cholate and the results presented in Tables 6 and 7, show

that the rates of hydrolysis, k' p and activation constants for the two substrates

become much closer for the two substrates in presence of cholate. Thus k 2 and/orI

k 3mst be increased by cholate. It is, of course, possible that one 6f these rate

constants is decreased by cholate provided that the other is sufficient3ly increased

to give rise to an overall increase in kapp, and an increase in the ratio k 2 3

might explain the convergenoe of the activation constants for TAM and TAU in presence

of cholate. This phenomenon could be also explained if cholate helps to remove the

bound alcohol from the specific site on the acylated trypuin, and permit its replace-

ment by water. Such a process would increase the rate of hydrolysis of the acylated

trypsin (i.e. increase k 3 ) and would explain, at least in pert, the observed accele-

ration of the hydrolyses of TAME, TA1U, and IA by oholate. Cholate may intervene

in these reactions in other ways, however, and the relative importance of the various

effects may depend on the particular substrate. Thus it will be seen (Table 7) that

when TAIM or T are used as substrates, cholate tends to decrease o and to make

more negative. Conversely, in the hydrolysis of MAE, although this also is

accelerated by cholate, addition of cholate increases WA and makes M less negative.

If it is assumed for the moment that deacylation of the acylated enzyme is the rate-

determining step, cholate apparently affects the structure of the aotivated state as

wenl as possibly influencing the binding of water or alcohol in the ground state.

sach perturbations of the activated state might include (1) alteration of the helical

content of the polypeptide chain, (2) alteration of the degree of solvent binding

(3) alteration of the number and distribution of charges. Alternatively, if the

foregoing postulate that the TMJ-trypsin and THMh-trypsin systems differ with regard

_iito the k 2 3 ratio is correct, the opposite effects of oholate on the apparent acti-

vation constants may reflect the differing weights of k 2 and k 3 in the composite

rate oonstnnt k 3(,Pp)for the two systems. It is interesting to note that increasing

cholate oonentrrtion above 0.002 M has little further effect on the kinetics of the

hydrolysis of UU0, unlike the onse of TAM, Yhere cholite oontinues to modify the

kinetics up to a concentration of 0.006 M. The effects of cholate on the hydrolysis

of the above substrates by trypsin is not due to general-bae catalysis, since acetate

ion w.s inactive.

Taue6.

Rate constonts at pH 8.4 for the trypsin-catalysed hydrolysis of derivatives

of r dnine and homoarinine in presence of cholate.

Rate constants (k'3 (11)) are quoted with standard devictions;

the number of determinations is given in parentheses.

J& holate (0() TAM V=TAM

15 0 57.98t 2.08(101,9.29 2.21(4) 2.286t 0.098(7 7.0,6±.174(4.)

15 2 69.20t 3.71 (4) 71.27± 2.39(4) 2.45*3 0.105(8 7.072±0.068(3)

15 4 81.661 2.97 (4 89.17-+ 7.11(4) 2.589± 0.062(w 6.406±o.169(4)is 6 92,5 33t 44t] 976&26( .&S2,tf.8 0.07L.(& 6.127-;,.,291[(jj

20 0 79.09± 2.21 (81 67.41+ 2.80(4) 3.00± 0.108(8 10.48 ±0.66 (4

20 2 90.21t 2.89 (4 98. 11 4.69(4) 3.10 0.065(7 9.767-0.310(4)

20 4 j04.5 - 8.1 (4 17.4 - 5.9 (4) 3.69t 0.228(4 9.52810.369(4)20 6 12J.5 7A (4 1.4 .3( 3.672+ 0.107(41 9.06co.i26(4)

23 0 85.72± 1.35 0+23 2 114.7 ± 3.3 (23 1+ 1330± 3.6 ()+

25 0 93.-0 ±8.73'%*17: 93.45 ±0.47 (3 4.04-1 ±0.212(8 14.38 10.62 (4)25 2 110.8 4. (15 124.6 - 0.8 (4. 4.411 !0.194(8 14.16 !1.27 (4)

25 . 129.1 + 7.7 05 152.3 ± 9.8 (4 4.766 !0.067(4 13 38 -0.-7 (4)25 6 I 6.9 65 66., 1 j. LAX 10 , J.75 .L6 (L)28 0 110.6 5.5 (6'

28 2 141.41.5 (5

28 4 166.4 5.4 (6'28 6 _8_.8 t _ (6+ ,

30 0 124.7 - 6.6 (10 123.8 ± 1.7 (3 5.331 10.231(8 .ei49 ± .37 (6)30 2 159.2 9.1 (4 162.4 ± 4.1 (4' 6.083 ±0.229(8 20.27 ±+-.60 (4)

30 4 189.5 3.7 (411%.7 ±-1,5 (). 6.437 !0-065( 19.86 ±1.26 (40 6 209,0 -12.6 (207,0 ! L.2 . 6,42 ..37 + ,

37 a 176.8 ± 7.5 (6)37 2 221,7 3.2 (5.

37 4 250.4 5.C (ii.

37 6 279.4±5.7 (04_________________

Activation oonstants at uH 8&L for the trypsin-oatalvsed hydrolysis of

derivativL of snirn and homoarainine in Dresenoe of oholate.

Aotivation Cholate () TAW Tku lu . BMConstant , ,.

0 9430-110 1,50-320 9810- 70 12510-350

3 (oais./mole) 2 9330 310 90 230 10280 360 11690 4504 8940 4.10 9010± 70 t0oo00 230 12750 ±306 8560 120 860 580 11060 610 12690 370

0 8830 110 9860 320 9220± 70 11920 350OR (oals./mole 2 8740 310 884.0 ±230 9680 360 11090 450

at 250C.) 4 8340±-i0 8420± 70 980 230 12160 3006 7970 ± 120 7860 580 10..70 610 12100 370

0 -i9.8 0.4 -16.4- 1.1 -24.8 ±0.2 -13.2 ±1.21-S (oals./mole/ 2 -19.7 ± 1.1 -19.3 ± 0.8 -23.0 ± 1.2 -16.o ± 1.5

°0 at 2500. 4 -20.7 ± 1.3 -20.3 ± 0.2 -22.5 0 0.8 -12.6 ± 1.0

6 -21.7 0.4 -22.0 1.9 -20.3± 2 -12.8 1.3

0 14749- 2 14762 2 16621 ±2 15856 10

oe (oals./aole 2 14610 ± 8 14591 - 2 16552 - 7 15886 ± 10at 250.: 4 I1509 -10 14474 - 16523 -4 15906- 7

6 14443 2 1419 7 16510 13 15929 7

The trypsin-catalysed t~drolysis of BAEE, unlike those of substrates bearing a

am*-toluene-2-sulhonjude group, m.s inhibited slightly by oholate. Since the

avnilable evideno. ineicates that acylation of trypsin by Rt- is so fPst thatI I

k 3 (app) = k 3 , it now appears that cholv-te might accelerate the acylation step and

slightly inhibit the deacylation stage.

We have foune thnt the apparent K. for the TIA-tzypuin system is ach lower

than the values quoted by Martin, Golubow, and axelrod (A. Al. n., 1959, 116

1718) and Ronwin (Bioch1m . y. igct 1959, j 326). The tcrer workers

determined % at ;H 8.0 end 250 in 0.1 X cnlcium chloride, 1hile Ronwip used either

veronal or succinrte buffer at 380 -nd determined % over a range of V1 values in

the presence of 0.0066 U calcium chloride. -c -rc unable to account Cor the lack

of agreement, although the conditions were not identical; in particular, we deter,-

mined % in the absence of buffers. Lt pH R.4 and 250, in prosence of 0.00 N

oalcium chloride, we find that the N (app) for the TAE-tzypsin system is 6.48 - 0.21

x id"%. In presence of 0.002 M cholate, the value is t.09 ± 0.08 x i0 5 . 7he value

of %(p)is sensitive to alterations in the valucs of all four rate constants, kipI k ik l, k2 , k,, Reference to equation 7 shows that an increase in k 3 would inorease

I1 -12 -

Twp) whl nices nk rl! ouddces h feto le

rations to k 2 depend on the relative mgnitude of k_1 and k 3" If k_, Ik y anincrase inwhilandincrease decreaseTheetioal, if k ( , the converse

would be true, but this woule" bo most unusual for an enzyme-catalysed reaction. It

in not possible to determine fron the present data which rate constants are affected

by oholate in the T1.M-trypain system.

Values of %(,Pp) for the TWJM-trypsin system under various conditions are

recorded in Table 8.

Tale8.

K( ) values for the TUH-trypsin system at 259.

(Values of K?(app) a quoted with stn&'C1C devir.tions; the

number of complete deturminations involving 6 - 8 runs is

given in parentheses).

PH Cholate oonc'n.(cU) i& K(ip)(moles/litre)

7.0 0 2.391 0.070 (3)

8.4 0 3.320 0.017 ()

8.4 2 2.51+3 ± 0.288 (1)

84 6 2.243 - 0.058 (2)

It will be seen that %(app) increases with pH over the range 7.0 - 84. Since

K, the dissociation constant of the group in the Michaelis complex is hiher than K0I I

it in likely that k 3 incrcnass considerably over this range, while k 2 is much le

sensitie. The incre.se in ) with increasing pH is probably attributable,

therefore, to the increase in k * The decrease in K,(,p) in presence of cholate,I I

on the other hand, is probably due to an increase in k2 The value of k3 probably

does not alter appreciably, as suggestee by the foregoing work, but a small chonge in

either direction is possible.

We were unable to study the trypsin-c'tri7ysed hydrolysis of Gj-toluene-2-

sulphonl-L-ornithine methyl ester (TOM) at ,H 8.4, in view of the latAnisation of

the substate, which is c:..t]ysed by hydroxyl ions (P-rt I). Trypain ws found to

cattlyse the hydrolysis of TE .t AH 7.0, however, with little oncomit-nt lact.-

mization. The %(,Pp) -ast be very high, since th,m was no dvyprture frcm first-

order kinetics with respect to substr te even when the substr-.te conoentrntion was

's "'gh is 5 x id . Pseudo-first-oreer r.t constatnts nre recorded in T.ble 9,

P..aaoa-ffrt-,orde- rate constant for the 1lstrvDin system at xE 7.0.

(lat constants are quoted with standard dbviati Ls; the number of

determinations is given in parentheses).

t 0 k (seo.-'/mole trypsin/i'tre).

No dcholate 2 di cholate

15 66.69 1 1.71 (,) 89.o6 - 2.18 (.)

20 97.83 3.06 (4) 123.08 - 2.57 (6)

25 133.02 ± 348 (4) 169.58 ± 2.78 (4)

30 193.39 ± 2.61 (6) 241.06 !11.90 (6)

In the presence of 0.002 M cholp-te, the apparent energy of aotivation is decrersed

from 12280 ± 290 oals./mole. to 11180 ± 260 cals./mole. It is not possible to decide

from the present &ta which step of the ensymic mechanism is affected by cholate. The

hydrolysis of N by trypsin does not appear to be completely stereospecifio. Using

the 5, ester, we found that the rr.te of alkali uptake in presence of trypsin was

peater than when the &-ester of half the molar concentration was used as substrate.

Moreover, when the B-ester w'.s used as substrate, alkali uptake continued beyond the

value representing 5CW hydrolysis. This was shomn not to be due to laotamization,

since a sample wns hydrolysed with typsin until 7).8% of the theoretical amount of

alknli required for complete hydrolysis had been used. 4t that point a S~remsen

titration indicated thAt only 8.6% of the total amount of ester had lctamised.

Finally, although good first-orCer plots coule be obtained from the hydrolysis of the

L-estwr in presence of trypsin, similr analysis of kinetic runs with the &[-ester

gave ourves, indicating that at least two reactions with different rate constants

were proceeding simultaneously. It is possible thrt the loss of complete stereo-

specificity is due to the shortness of the sido-chain of ornithine compared with

arginine rnd lysine. Attachment of TO to a specific site on the eneyme moleoule

by the protonated amino group my be weakened. Further shortening of the side-oh-An

might preclude 1jW attachment to the specific site and this presumably explains the

failure of trypsin to catalyse the hydrolysis of A IE.

PAR m. sTMMS CP M r x u

Thrombin in a proteolytic enzyme which is responsible for Ahe oonversion of

fibrinogen into fibrin during blood-clotting; it also displays esaterase activity

tow rds substrates like TAS. Inhibition of thrombin by di-isprofl phosphovo-

fluoridte, followed by partial hydrolysis, has revealed that this ensyme, le

bypsin, chymotrypsin, elastase, and cholinesterase, has the amino-acid sequence

- gly.asp.ser.V - at the active centre. Unfortunately, t1nombin has not been

purified as completely as trypsin or ckmotrypsin, and it is necessary to define a

suitable unit of activity. The literature abounds with different units for expres-

sing thrombin activity, and we have selected that due to Shrempreis and 8cheraga

(&. .20., 1957, 22, I ) as the most suitable for our purpose. One

thrombin unit is defined as that amount of enzyme required to liberate 0.1 poole of

acid per manute from I al. of 0.1M TAU at pH 8 in 0.15 M I at 2!P. We were

able to confirm the observation of 1hrenpreis and Scheraga that thrombin solutions

at pH 7 and 0eC retain their esterase activity for at least nine days. On the

other hand Ronwin (CanaL J. Bio ham. Physiol., 1957, Zo 743) reports that thrombin,

dissolved in glyoerol-mater (i : i) or isotonic saline, becomes more active as an

eaterase during storage at 0-4o. Using BAEZ (5 x 10)M) as substrate, we found

that zero-order kinetics were obeyed and the velocity was proportional to the

enzyme concentration.

The throbin-oatalysed hydrolysis of TAU is markedly dependent en ionic

strength. Ronwin (Canad. J. Biochem. Ptwasol., 1957, 3, 743) has reported that

increase of ionic strength inhibited this reaction, but since he used buffers as

well, the results are difficult to interpret. Our results (Table 10) were obtained

in the gbeence of buffers, and it can be clearly seen that k3 (app) decreases

asymptotically with increasing strength to a limiting value which depends on the

cation. Our results do not afford a linear relationship between log k and

where p is the ionic strength, as might have been expected. Scheraga and

2brenpreis . tM. 2=. Bichmir, Venna ergamon Press,

Vol. X, p.212) have briefly reported that sodium chloride inhibits the hydrolysis

of TiME by thrombin as the ionic strength is increased. Their results qualita-

tively resemble ours and afford no linear relationship between log k and , .

A possible explanation for the complex connexion between log k and p may be the

presence of more than one esterase activity in the thrombin preparations.

Tbe0.

Effect of ionic strength on k' for the thrombin-oata~lsed

! hvg Iya ofTAKE at yH 8.1& and 2500.

k 3 (aPP) values are quoted with standard deviations; the number

of -',.-trminations is given in parentheses.

Cne'.. of salt (M) 10A31 (app)(mo3eslitelmn.lsheraga uit/i~tre).

0.00 18.071 - 0.076 (5) 18.071 ± 0.076 (5)

0.05 9.082 0.663 (3) i3.1i. + 0.381 (3)

0.10 6.7,54 0.1*31 (4) 12.361 ± 0.238 (2)

0.20 5.114 ± 0.808 (2) 10.802 ± 0.751 (2)

0.30 4.289 4. 0.10 (2) 8.832 ± 0.125 (2)

0.40 3.880 - 0.287 (2) 8.809 ±. 0166 (2)

Surprisingly, the rate of hyerolysis of BAM by thrombin was insensitive to

the ionic strength when sodium or potassium chloride was added to the system. A

possible explanation of the inhibition of hydrolysis of TAME by thrombin with in-

creasing ionic strength is the existence of a mechanism which involves the approach

of ions of unlike charges. Since TAME has a positive charge on the guanidine group,

it sight be argued that a negative charge exists at the active centre of thrombia.

Baoh a charge does exist on the A- 002- group of the aspartyl residue, adjacent to

the serine residue which is phosphorylated by di-isopropyl phosphorofluoridate.

This explanation is weakened, however, by the insensitivity of the BA3-thrombin

and TAME-trypsin systems to change of ionic strength. On the other hand, it is

possible, as outlined below, that the acylation step controlled by ks contributes

to ki (app) in the TAME-thrombin system, but not in the BABB-system. In this

event, the acylation step only could be sensitive to change of ionic strength, in

which case, it could be an indication that a negatively charged group is the

nuoleophile which attacks the oarboiyl-carbon atom in the substrate. Further

evidence must be obtained before this point can be established. It is puzzling

that the TAM-trypsin system is insensitive to change of ionic strength when other

evidence indicates that the acylation step is partially responsible for the observed

value of k' It is of interest to recall that the velocities of certain3 (app)-hydrolyses catalysed by chymotrypsin and ficin are increased as the ionic strength is

inreased (Shine and Niemann, J. Amer. Chem. Soc., 1955, Z 4275,; Martin and

Niemarz, J. Imer. Chem. Soo., 1958, _ 1481; Hamond and Gutfreund, lodhe. J.,

059, 2L X9)-

-16.-

We have determined k (app) for the hydrolysis of a range of esters in presence

of thrombin at pH 8.4 and 25 0 C. These include TAME, TAN!, TAM, RA, DAME, --

toluene-2-sulphorWlj--arginine -propyl esttcr (TAPE), and -J-touene-2sulphory:L..-

lysine methyl ester (TMUE). The results are presented in Table 11.

Table 1i.

k/ ) for the hydrolysis of various substrates by thrombin at pH 8.4 and.

2500 in 0.1 M sodium chloride solution.

Valuestctkl 'app) are quoted with standard deviations; the number of deter-

minations is given in parentheses.

Substate iO5k'Substrte I A app)(moleslitre/mn./scheraaa unit/litre).

TAME 6.754 0.431 (4)

TAU 2.988 - 0.051(10)

TM 2.611 ± o.o56 (6)

T6.538 0.088 (6)

U 10.430 o.6o (6)

BAE 3.233 ± 0.071 (5)

BARE3.246 ±0.013(05)

it will be noticed that T.AQE is hydrolysed considerably morm rapidly than TAM Or

TAPE, and it is intereting tbat this order is the opposite of that expected in the

ncn-ermymic alkaline hydrolysis of esters. Ls wrs found in the case of trypaim,

esters ontaining a a-1-toluene-R-sulphonamide group do not hydrolyse at the saem

rate. Henoe, we may conclude that for thesc substrrtes, deacylation of an aq:L-

thrombin is not uniquely rnte-determining; the acylation step governed by 14 makes

a significant contribution to k' ., On the other hand BAME and BM are3 'app)bydrolysed by thrombin at indistinguishable rates, and it may be concluded that

deaoylation is rate-determininp, i~e k I Ic, I'* The foregoing observatiora3 ap) 3are thus similar to but rather more striking than those found using tr'ypsin. On

further point may be noted; T and TAM are hydrolysed at about the same rate by

thrombin. The distanct between the charged group in the side-chain and the ester-

carboryl group is almost identical for the two substrates ihen the .oleaules eve in

their energetically most favoured conformations. '5e have indicated in the studies

on trypain that the length of the side-chain of the substrate is important in cleter-

mining specificity, and the same seems to be true in the case of thrcmino In

preliminary experiments, m have found that TOM is slowly hydrolysed by throdbis

-17-

and the apparent % is high, as in the oae of trypsin. TRAM, on the other hand,

is not appreoably bydro3.ysed by thrombin, and in this respect contrasts sharply with

trpsil.

In the previous Final Teohnioal Report we recorded the results of a limited

stu43 of the effect of temperature variation on k' (aPP) for the TAM-thrombin my&-

tern. The results available at that time indicated that an Arrhenius type of plot

was linear. More extensive measurements using both TAME and BABE as substrates

have since shown that plots of log k', (app) against I/T have a marked curvature

(Tables 12 and 13, Figs. I and 2). We are thus unable to calculate activation con-

el -1 for tbrombin-eatalysed reactions. It is likely that this abnormal behaviot

results from the presence of more than one type of esterase activity in the thrombin

preparation. This is substantiated by the very recent iselation by Maroiniak and

Seegers (91 . & Bio .-zwol., 1962, J& 597) of two fractions possessing

esterase activity from highly purified thrombin. Ronwin (goa. g. Roah. Physiol.,

1957, 2, 743) found that zero-order kinetics in the TAME-thrombin system were not

obeyed at 380 when small concentrations of enzyme were employed, but normal behaviour

was found in experiments when relatively large amounts of thrombin were used.

Neither we nor Sherry and Troll (J. Biol. Chem., 1954, 2, 95) have observed this

pheInon.

Effeot of tewerature n kj (app) for the TafL-t bin avstem at

m1 8Ja. alone and in the uresenoe of cholate.

Values of k3 (app) are quoted with standard deviations; the rum-

ber of determinations is given in parentheses.

No cholate P Idi cholate

15 4.826 097 '3 6.786 1 0.252 317.5 5.058 -0.8 4 7.289 0.095 420 5.869 ± 0.031 '4 8.231 0.35322.5 5.965 - 0.124 4 8.207 ; 0.15725 6.754 - 0.431 4 9.875 ; 0.337 '530 9.486 0.517 41 13.533 ; 0.(00 437 13.499 - 0.015 4 20.512 0.056 1

-LOG~, -

x z

00

300

0 4 4

-LOG,,ks .

* CA

100

C3.-r"

Effetof teme on k _ (app) for the EAMI-thrombin asvtem

at r1 8.Ji. alone and in the ioresence of oholate.

Values of k Iap are quoted with standard deviations; the

number of determinations is given in parentheses.

tera 1o 5km (app) (moleqitre/min. cherage unit/litre)

Vj No cholate 2m( cholate &aM cholate

1o 1.283 0 0.023 (4) 1-043 ±- o.04 (4)

15 1.627 0.037 (3) 1.197 0.051 () 1.116 0.055 (3) 1.047 ±0.053 (3)

17.5 1.695 ± o.017 (4) 1.279 0 o.07 ()

20 2.10 o.061 (4) 1.703± .011 ()

22.5 2.75+ 0.013 (4) 1.773 -.065 (3)

25 3.24.6 ± o.M3(15) 2.370 o.o88 (3) 1.798 ± 0.059 (3) 1.603 ± 0.069 (3)27.5 3.-7 - 0.025 (4) 2.388 0.096 (3) 1.826 ± 0.067 (3) 1.690 ± 0.1O (3)

30 .212 ± 0.051 (3) 2.926 ± 0.091 (5) 2.457 ± 0.093 (5) 2.217 - 0.065 (5)

37 8,8937 ±0.021 0) 5.236 ± 0.011 (3) 1.1 05 o.o2o () 1. 519 o.016 (4)

Ronwin (M.him " j os. . A21 1959, j 326) has studied the effect of pH-

variation on k for the TAME-thrombin system and has reported that there are

two dissociating groups in the ES complex, and three in the free enzyme. In con-

treat, e detected only one dissociable group, P%app) = 6.46 t 0.06, in the TAME-

trypsin system by measuring k(ap) at various pH values at high substrate con-

centration. This could bu an imidasole group, but it is likely that the observed

pK, lies between those of the dissociable group an the HS complex and that on the

acylated enzyme, since it appears from the results described above tlmt mylation

is partly rate-determining.

The thrombin-catalysed hydrolyses of substrates such as TAME, TmL, 7194 and

MME, Which contain a L--toluene-2-sulphonamide group are accelerated by cholate

and even more by glycocholate. The thrombin-catalysec hydrolysis of BARM, on the

other hand, is markedly inhibited by both cholate and glyoocholate. The inhibition

of this system is more pronounced than in the case of the tt- in-oatalyaed

hydrolysis of BAR. The kinetic modification by bile salts of both trypsin- and

thrombin-catalysed reactions appears to operate by a similar mechanism; acylaticn

is accelerated while deacylation is retarded. The results are recorded in Tables

12, 13, and 14. There is a quantitative difference between the effects of cholate

and glycocholate. The former, from its effect on the byholyais of BAIE, appears

to be the more powerful inhibitor of the deacylation step. Glyoocholate, on the

other hand, is the more effectiv nccelerator of the hydrolysis of TAME. This ic

-49-consonant with the postulates that both k2 and k 3 contribute to the overall rate or

hydrolysis of TIJ and that bile salts aoelerate the acylation step governed by%

osand ihibit the de-tcylation step gover n e d by k 3 t

The effect of oholate and a1vcoocholate on k3(app) for throni

catalvsed hycrolyses of derivatives of arinine and lvsine at M 8.1 an, 250.

Values of k36rPP) ae quoted with standard deviations; the

number of determinations is given in parentheses.

Conc'nBile Salt (MM) 10 5 k 3 (,p) (mole /litre/min./Scherga unit/litre).

_______TAC ________ _______ BAM

None 0 6.754 - 0.431(4) 2.988 + 0.051(10) 10.430 0 0.60(6)13.246 - 0.013(15

2 9.875 4 0.337(5) 4.539 ± 0.062 (3) 14.17 0.0(6) 2.370 - 0.088 (3,

Cholate 4 14.94 ± 0.16(3) 1.798 4 0.058 (3:

6 15.96 0 o.63(3) 1.603 ± 0.069 (0

2 13.98 ± 0.22 (5) 15.29 0 0.30(4) 2.326 0 0.025 (4'

Glycocholate 4 15.78 ± 0.+9 (6) 17.80 0.46(3) 2.202 - 0.029 (4',

6 6.09 .0.19 (4) 19.29 ± 0.27(4) 1.965 , 0.140 (3

We have also determined the npptrent K4 for the BAZE- and TAME-throubin systems

alone and in the presence of cholate and glycochol: te. The values of N(app) in the

absence of bile salts were sufficiently high to be determined by the orthodox

technique. In presence of bile salts, however, the values of K(,p ) were depressed

to an extent that -nde it necessary to measure velocities during the course of single

runs by the process of stctistically fitting orthoFonal polynomials to the experi-

mental pH-stat traces -nd differentiating the polynomials at various stages during

the reactions. This method c-n only be used if the products of hydrolysis are not

inhibitory. We showed in sep rate experiments thrt adition of a-n-toluene- R -

sulphonyl-L-erginine to the T1M-thrombin systcm or of a-l-benzoyl-k-orginine to the

BAE thrombin system did not oroduce any detectable alteration in velocity. The

method is liable to fairly considernble error, but the effect of bile salts is

sufficiently Dronounced for certain trends to be seen. The results are presented

in Table 15.

-20-

Tale 15.

AA (app) in the -and !h-throtio_ systems at DH 8.. -d 250

Mean values of %(app) are quoted with standard deviations; the

number of determinations is given in parentheses.

Bile Salt i o0% (app) (oles/itre)

None 1,61 - 0.39 (5) 3.21 ± 0.78 (7)

Cholate (2rr1) 0.6o ± o.1o (6) 0.50 ± 0.23 (6)

Glycholate (2ii) 0.74 - 0.14 (5) 0.54 - 0.26 (5)

I S

The effect on %(ap) of alterations to k2 nd k; is analysed in Part 17. An! aj2

increase in 2 or a decrease in ky both of which appear to result from addition of

bile salts, lead to a decrease in (ap). The observation that the % (app) for the

TANE-thrombin system is more sensitive than that for the BAZ-thrombin system in

presence of cholate is consistent with the view that k 2 )> k 3 in the latter case.

A few experiments have been carried out to determine the effect of the non-ionio

detergents Tween 20, Tween 40, and Tween 80 on thrombin-catalysed reactions. The

hydrolysis of BAEE is slightly accelerated, hile that of TAME is considerably

accelerated. In the light of the foregoing discussion, it appears that the non-

ionic detergents slightly increase k3 and effect a considerable increase in k 2. The

results are given in Table 16.

Table -16,ffect of non-ionic detergents on k3(app) for the th'ombin-catalysed

bwdrolvsis of TM. and AM at nH 8.4 and 250.

Values of k;(aPP) are quoted w ith standard deviations; the rAniber

of determinations is given in parentheses.

Detergent ConW,,t4ron ik3 aP (mole s/litre/isin./cheraga uit/*itre).

TAM BAE

None 0 6.754 ± 0.431 (4) 3.26 0 0.013 (15)

Tween 20 0.3 9.422 0.140 (3) 3.667 - 0.096 (4)

1.2 11.026 0.486 (3)

Tween 40 0.3 9.219 ± 0.331 (4)

1.2 9.59 0.097 (4) 3.- 0.062 (3)

Tween 80 0.3 11.603 - 0.301 (5) 3.920 ± 0.116 (4)

-21-

Prom the foregoing work, it is evident that trypsin and thrombin closely resemble

one another. Thrombin, however, does appear to be somersat more specific (see

Scheraga and Laskowski, AAX. Protein Chem., 1957, Z i), since it has virtually no

action on TRW. The conversion of fibrinogen into fibrin is a further example of

the greater specificity of thrombin compared with trypsin. Although fibrin is not a

substrate for thrombin, except perhaps in presence of high enzyme concentrations

(Quest and Ware, Science 1950, 112, 21), trypsin is capable of effecting considerable

degradation involving the cleavage of peptide bonds. Both thrombin and trypsin appear

to octalyse the hydrolysis of synthetic substrates by a three-step mechanism, and, for

a given substrate, the relative importance of each step in determining the overall

kinetics is very similar. The behaviour of kinetic modifiers such as cholate appears

to be qualitatively similar with both enzymes; the step governed by 4 is accelerated

and that controlled by k' is slightly retarded. If this theory can be confirmed by.3

further experiments using other substrntes, it is possible that such kinetic modifiers

will be useful in elucidating the mechanism of enzyme-catalysed processem. It is not

possible yet to assess with certainty the importance of anionic surface-active Wients

such as bile salts or ohospholipids on biological processes such as protein digestion

and blood-clotting, which depend on trypsin nnd thrombin. The kinetic mechanism of

those reactions described in this report appears to be more complex than at first

seemed to be the case. "se have not had time to study the effect of cholate and glyoo-

cholate on more complex reactions such as the conversion of fibrinogen into fibrin,

have we been able to study the kinetics of renctions catalysed by pla.a. Clearly, it

would be of considerable interest to do this. We would have liked to have studied

the effect of bile salts on the pre-steady state using substrates such as p-nitrphenyl

acetate, but delays in obtaining commercial equipment precluded this. It is hoped,

however, that the authors will be in a position to tackle some of these problems in

the near future.

P'RSOHW TnI 'r AN E

Mr. N. J. Baines worked on this contract from its commencement until the end of

the second quarter. Mr. E. F. Curragh has worked on this contract throughout the

whole period. Both men have devoted their complete attention to the problem during

the time that they have been paid under the contract.

The cost of chomicals and of preparing reports has exceeded the original estimate

of Z79.